Dehydrogenaza oktopinowa z Pecten maximus: mechanizm stanu stacjonarnego.

Mechanizm kinetyczny reakcji w stanie stacjonarnym katalizowanej przez dehydrogenazę oktopinową [N2-(1-karboksyetylo)-L-arginina:NAD+ oksydoreduktaza] badano przy pH 6,9 i 9,2 w badaniach hamowania substratu, hamowania analogu i hamowania produktu . W kierunku syntezy oktopiny wzory inhibicji w obecności deltaguanidowalerianu i propionianu pokazują, że NADH wiąże się najpierw z enzymem, a następnie z L-argininą i pirogronianem, które wiążą się losowo.

W kierunku utleniania oktopiny wzorce substratów pokazują, że NAD wiąże się z enzymem przed oktopiną w szybki sposób w stanie równowagi, a wzorce inhibicji produktu pokazują, że produkty L-arginina i pirogronian są uwalniane w sposób losowy. Wykazano, że podwójne, synergistyczne hamowanie substratu przez L-argininę i pirogronian jest spowodowane wiązaniem (hipotetycznie iminy) z wolnym enzymem i kompleksem enzym-NAD. Ponadto wykazano alternatywny szlak o mniejszym znaczeniu, który obejmuje kompleks enzym-NADH-oktopina i kompleks enzym-oktopina

Dehydrogenaza oktopinowa (OcDH) z mięśnia przywodziciela przegrzebka olbrzymiego, Pecten maximus, katalizuje zależną od NADH, redukcyjną kondensację L-argininy i pirogronianu do oktopiny, NAD(+) i wody podczas pływania ucieczkowego i/lub późniejszej regeneracji.

  • Niedawno rozwiązano strukturę OcDH i zaproponowano mechanizm reakcji, który implikował uporządkowane wiązanie NADH, L-argininy i wreszcie pirogronianu. W tym przypadku kolejność wiązania substratu, a także leżące u podstaw zmiany konformacyjne zostały zbadane za pomocą NMR, potwierdzając model wyprowadzony ze struktur krystalicznych. Ponadto określono strukturę krystaliczną kompleksu OcDH/NADH/agmatyna, co sugeruje kluczową rolę łańcucha bocznego L-argininy w katalizie białek.
  • W ten sposób można teraz szczegółowo opisać kolejność wiązania substratu z OcDH, a także sygnały molekularne zaangażowane w tworzenie oktopiny.
  • Dehydrogenaza oktopinowa [N(2)-(D-1-karboksyetylo)-L-arginina:NAD(+) oksydoreduktaza] (OcDH) z mięśnia przywodziciela przegrzebka wielkiego Pecten maximus katalizuje redukcyjną kondensację l-argininy i pirogronianu do oktopina podczas pływania ucieczkowego.
  • Enzym ten, będący prototypem dehydrogenaz opinowych (OpDH), utlenia NADH pochodzący z glikolizy do NAD(+), podtrzymując w ten sposób beztlenowe dostarczanie ATP podczas krótkich okresów wzmożonej aktywności mięśni. W przeciwieństwie do niektórych innych OpDH, OcDH wykorzystuje tylko l-argininę jako substrat aminokwasowy.
  • W tym miejscu opisujemy struktury krystaliczne OcDH w kompleksie z NADH i kompleksami binarnymi NADH/l-arginina i NADH/pirogronian, dostarczając szczegółowych informacji na temat zasad rozpoznawania substratu, wiązania ligandów i mechanizmu reakcji.
  • OcDH wiąże swoje substraty poprzez połączenie sił elektrostatycznych i doboru wielkości, co gwarantuje, że kataliza OcDH przebiega z selektywnością substratową i stereoselektywnością, dając początek drugiemu centrum chiralnemu i wykorzystując mechanizm „linii molekularnej”.

cDNA dla dehydrogenazy oktopinowej (ODH) z mięśnia przywodziciela przegrzebka olbrzymiego, Pecten maximus, sklonowano przy użyciu 5′- i 3′-RACE. cDNA zawiera ORF o długości 1197 nukleotydów, a wydedukowana sekwencja aminokwasowa koduje białko o długości 399 aminokwasów.

ODH ulegał heterologicznej ekspresji w Escherichia coli z C-końcowym penta-tagiem His. ODH-5His oczyszczono do jednorodności stosując chromatografię powinowactwa z chelatem metalu i filtrację żelową Sephadex G-100. Rekombinowany ODH miał właściwości kinetyczne podobne do ODH typu dzikiego wyizolowanego z mięśnia przywodziciela przegrzebka. Do wyjaśnienia udziału kilku reszt aminokwasowych w reakcji katalizowanej przez ODH zastosowano ukierunkowaną mutagenezę. Cys148, która jest konserwowana we wszystkich znanych do tej pory dehydrogenazach opinowych, została przekształcona w serynę lub alaninę, co pokazuje, że ta pozostałość nie jest z natury istotna dla katalizy.

His212, Arg324 i Asp329, które są również konserwowane we wszystkich znanych sekwencjach dehydrogenazy opinowej, poddano ukierunkowanej mutagenezie. Modyfikacja tych reszt ujawniła ich znaczenie dla aktywności katalitycznej enzymu. Konwersja każdej z tych reszt do alaniny spowodowała silny wzrost wartości K(m) i spadek wartości k(cat) dla pirogronianu i L-argininy, ale miała niewielki wpływ na wartości K(m) i k(cat) dla NADH .

Zakładając podobną strukturę ODH w porównaniu z jedyną dostępną strukturą bakteryjnej dehydrogenazy opinowej, te trzy aminokwasy mogą funkcjonować jako triada katalityczna w ODH, podobna do tej występującej w dehydrogenazie mleczanowej lub dehydrogenazie jabłczanowej. Grupa karboksylowa pirogronianu jest następnie stabilizowana przez Arg324. Oprócz orientowania substratu, His212 będzie działał jako katalizator kwasowo-zasadowy, oddając proton grupie karbonylowej pirogronianu. Kwasowość tej histydyny dodatkowo zwiększa bliskość Asp32.

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MBS1339775-002mgYeast MyBiosource 0.02mg(Yeast) 820 EUR

Recombinant Elephas maximus Hemoglobin subunit delta (HBD)

MBS1339775-01mgEColi MyBiosource 0.1mg(E-Coli) 760 EUR

Recombinant Elephas maximus Hemoglobin subunit delta (HBD)

MBS1339775-01mgYeast MyBiosource 0.1mg(Yeast) 960 EUR

Recombinant Elephas maximus Hemoglobin subunit alpha (HBA)

MBS1086614-002mgBaculovirus MyBiosource 0.02mg(Baculovirus) 1065 EUR

Recombinant Elephas maximus Hemoglobin subunit alpha (HBA)

MBS1086614-002mgEColi MyBiosource 0.02mg(E-Coli) 650 EUR

Recombinant Elephas maximus Hemoglobin subunit alpha (HBA)

MBS1086614-002mgYeast MyBiosource 0.02mg(Yeast) 815 EUR

Recombinant Elephas maximus Hemoglobin subunit alpha (HBA)

MBS1086614-01mgEColi MyBiosource 0.1mg(E-Coli) 750 EUR

Recombinant Elephas maximus Hemoglobin subunit alpha (HBA)

MBS1086614-01mgYeast MyBiosource 0.1mg(Yeast) 955 EUR

Recombinant Elephas maximus Hemoglobin subunit gamma (HBG)

MBS1474908-002mgBaculovirus MyBiosource 0.02mg(Baculovirus) 1070 EUR

Recombinant Elephas maximus Hemoglobin subunit gamma (HBG)

MBS1474908-002mgEColi MyBiosource 0.02mg(E-Coli) 655 EUR

Recombinant Elephas maximus Hemoglobin subunit gamma (HBG)

MBS1474908-002mgYeast MyBiosource 0.02mg(Yeast) 820 EUR

Recombinant Elephas maximus Hemoglobin subunit gamma (HBG)

MBS1474908-01mgEColi MyBiosource 0.1mg(E-Coli) 760 EUR

Recombinant Elephas maximus Hemoglobin subunit gamma (HBG)

MBS1474908-01mgYeast MyBiosource 0.1mg(Yeast) 960 EUR

Recombinant Elephas maximus Cytochrome b (MT-CYB), partial

MBS1156768-1mgEColi MyBiosource 1mg(E-Coli) 2025 EUR

Recombinant Elephas maximus Cytochrome b (MT-CYB), partial

MBS1156768-1mgYeast MyBiosource 1mg(Yeast) 2470 EUR

Recombinant Elephas maximus ATP synthase subunit a (MT-ATP6)

MBS7021411-002mg MyBiosource 0.02mg 1515 EUR

Recombinant Elephas maximus ATP synthase subunit a (MT-ATP6)

MBS7021411-01mg MyBiosource 0.1mg 2475 EUR

Recombinant Elephas maximus ATP synthase subunit a (MT-ATP6)

MBS7021411-5x01mg MyBiosource 5x0.1mg 11090 EUR

Recombinant Elephas maximus Alpha-2B adrenergic receptor (ADRA2B)

MBS1206227-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus Alpha-2B adrenergic receptor (ADRA2B)

MBS7007308-002mg MyBiosource 0.02mg 1705 EUR

Recombinant Elephas maximus Alpha-2B adrenergic receptor (ADRA2B)

MBS7007308-01mg MyBiosource 0.1mg 2665 EUR

Recombinant Elephas maximus Alpha-2B adrenergic receptor (ADRA2B)

MBS7007308-5x01mg MyBiosource 5x0.1mg 11945 EUR

Recombinant Dipetalogaster maximus Serine protease inhibitor dipetalogastin

MBS1131148-002mgBaculovirus MyBiosource 0.02mg(Baculovirus) 1140 EUR

Recombinant Dipetalogaster maximus Serine protease inhibitor dipetalogastin

MBS1131148-002mgEColi MyBiosource 0.02mg(E-Coli) 725 EUR

Recombinant Dipetalogaster maximus Serine protease inhibitor dipetalogastin

MBS1131148-002mgYeast MyBiosource 0.02mg(Yeast) 890 EUR

Recombinant Dipetalogaster maximus Serine protease inhibitor dipetalogastin

MBS1131148-01mgEColi MyBiosource 0.1mg(E-Coli) 870 EUR

Recombinant Dipetalogaster maximus Serine protease inhibitor dipetalogastin

MBS1131148-01mgYeast MyBiosource 0.1mg(Yeast) 1045 EUR

Recombinant Elephas maximus ATP synthase protein 8 (MT-ATP8), partial

MBS1427213-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 925 EUR

Recombinant Elephas maximus ATP synthase protein 8 (MT-ATP8), partial

MBS1427213-005mgMammalianCell MyBiosource 0.05mg(Mammalian-Cell) 1150 EUR

Recombinant Elephas maximus ATP synthase protein 8 (MT-ATP8), partial

MBS1427213-05mgEColi MyBiosource 0.5mg(E-Coli) 925 EUR

Recombinant Elephas maximus ATP synthase protein 8 (MT-ATP8), partial

MBS1427213-05mgYeast MyBiosource 0.5mg(Yeast) 1150 EUR

Recombinant Elephas maximus ATP synthase protein 8 (MT-ATP8), partial

MBS1427213-1mgEColi MyBiosource 1mg(E-Coli) 1255 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 2 (MT-CO2)

MBS7021548-002mg MyBiosource 0.02mg 1520 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 2 (MT-CO2)

MBS7021548-01mg MyBiosource 0.1mg 2480 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 2 (MT-CO2)

MBS7021548-5x01mg MyBiosource 5x0.1mg 11115 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 3 (MT-CO3)

MBS7021700-002mg MyBiosource 0.02mg 1560 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 3 (MT-CO3)

MBS7021700-01mg MyBiosource 0.1mg 2520 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 3 (MT-CO3)

MBS7021700-5x01mg MyBiosource 5x0.1mg 11295 EUR

Recombinant Elephas maximus ATP synthase subunit a (MT-ATP6), partial

MBS7076700-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus Alpha-2B adrenergic receptor (ADRA2B), partial

MBS7053638-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)

MBS7022026-002mg MyBiosource 0.02mg 1385 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)

MBS7022026-01mg MyBiosource 0.1mg 2350 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)

MBS7022026-5x01mg MyBiosource 5x0.1mg 10520 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 6 (MT-ND6)

MBS7022427-002mg MyBiosource 0.02mg 1455 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 6 (MT-ND6)

MBS7022427-01mg MyBiosource 0.1mg 2420 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 6 (MT-ND6)

MBS7022427-5x01mg MyBiosource 5x0.1mg 10835 EUR

Recombinant Elephas maximus Cytochrome c oxidase subunit 3 (MT-CO3), partial

MBS7075967-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus Cytochrome c oxidase subunit 1 (MT-CO1), partial

MBS7076485-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus Cytochrome c oxidase subunit 2 (MT-CO2), partial

MBS7076591-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

MBS7022196-002mg MyBiosource 0.02mg 1360 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

MBS7022196-01mg MyBiosource 0.1mg 2325 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L)

MBS7022196-5x01mg MyBiosource 5x0.1mg 10425 EUR

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 3 (MT-ND3), partial

MBS7076060-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 4 (MT-ND4), partial

MBS7076170-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 1 (MT-ND1), partial

MBS7076171-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 6 (MT-ND6), partial

MBS7076383-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 2 (MT-ND2), partial

MBS7076384-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 4L (MT-ND4L), partial

MBS7076484-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Elephas maximus NADH-ubiquinone oxidoreductase chain 5 (MT-ND5), partial

MBS7076590-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Priodontes maximus NADH-ubiquinone oxidoreductase chain 1 (MT-ND1), partial

MBS7088739-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Octopus vulgaris Octopressin receptor (OPR)

MBS7024619-002mg MyBiosource 0.02mg 1750 EUR

Recombinant Octopus vulgaris Octopressin receptor (OPR)

MBS7024619-01mg MyBiosource 0.1mg 2705 EUR

Recombinant Octopus vulgaris Octopressin receptor (OPR)

MBS7024619-5x01mg MyBiosource 5x0.1mg 12130 EUR

Recombinant Octopus vulgaris Octopressin receptor (OPR), partial

MBS7083973-INQUIRE MyBiosource INQUIRE Ask for price

Recombinant Bombyx mori Octopamine receptor

MBS7036759-002mg MyBiosource 0.02mg 1825 EUR

Recombinant Bombyx mori Octopamine receptor

MBS7036759-01mg MyBiosource 0.1mg 2755 EUR

Recombinant Bombyx mori Octopamine receptor

MBS7036759-5x01mg MyBiosource 5x0.1mg 12365 EUR

Recombinant Octopus vulgaris Cephalotocin

MBS1241542-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 870 EUR

Recombinant Octopus vulgaris Cephalotocin

MBS1241542-005mgEColi MyBiosource 0.05mg(E-Coli) 485 EUR

Recombinant Octopus vulgaris Cephalotocin

MBS1241542-005mgYeast MyBiosource 0.05mg(Yeast) 705 EUR

Recombinant Octopus vulgaris Cephalotocin

MBS1241542-02mgEColi MyBiosource 0.2mg(E-Coli) 635 EUR

Recombinant Octopus vulgaris Cephalotocin

MBS1241542-05mgEColi MyBiosource 0.5mg(E-Coli) 695 EUR

Recombinant Octopus vulgaris Tachykinin-1

MBS7092685-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 875 EUR

Recombinant Octopus vulgaris Tachykinin-1

MBS7092685-005mgEColi MyBiosource 0.05mg(E-Coli) 490 EUR

Recombinant Octopus vulgaris Tachykinin-1

MBS7092685-005mgYeast MyBiosource 0.05mg(Yeast) 695 EUR

Recombinant Octopus vulgaris Tachykinin-1

MBS7092685-02mgEColi MyBiosource 0.2mg(E-Coli) 640 EUR

Recombinant Octopus vulgaris Tachykinin-1

MBS7092685-05mgEColi MyBiosource 0.5mg(E-Coli) 695 EUR

Recombinant Octopus vulgaris Tachykinin-2

MBS7093717-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 875 EUR

Recombinant Octopus vulgaris Tachykinin-2

MBS7093717-005mgEColi MyBiosource 0.05mg(E-Coli) 490 EUR

Recombinant Octopus vulgaris Tachykinin-2

MBS7093717-005mgYeast MyBiosource 0.05mg(Yeast) 695 EUR

Recombinant Octopus vulgaris Tachykinin-2

MBS7093717-02mgEColi MyBiosource 0.2mg(E-Coli) 640 EUR

Recombinant Octopus vulgaris Tachykinin-2

MBS7093717-05mgEColi MyBiosource 0.5mg(E-Coli) 695 EUR

Recombinant Octopus vulgaris Sperm protamine P3

MBS1143278-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 885 EUR

Recombinant Octopus vulgaris Sperm protamine P3

MBS1143278-005mgEColi MyBiosource 0.05mg(E-Coli) 505 EUR

Recombinant Octopus vulgaris Sperm protamine P3

MBS1143278-005mgYeast MyBiosource 0.05mg(Yeast) 705 EUR

Recombinant Octopus vulgaris Sperm protamine P3

MBS1143278-02mgEColi MyBiosource 0.2mg(E-Coli) 660 EUR

Recombinant Octopus vulgaris Sperm protamine P3

MBS1143278-05mgEColi MyBiosource 0.5mg(E-Coli) 705 EUR

Recombinant Octopus vulgaris Sperm protamine P1

MBS1167070-002mgBaculovirus MyBiosource 0.02mg(Baculovirus) 980 EUR

Recombinant Octopus vulgaris Sperm protamine P1

MBS1167070-002mgEColi MyBiosource 0.02mg(E-Coli) 540 EUR

Recombinant Octopus vulgaris Sperm protamine P1

MBS1167070-002mgYeast MyBiosource 0.02mg(Yeast) 735 EUR

Recombinant Octopus vulgaris Sperm protamine P1

MBS1167070-01mgEColi MyBiosource 0.1mg(E-Coli) 645 EUR

Recombinant Octopus vulgaris Sperm protamine P1

MBS1167070-01mgYeast MyBiosource 0.1mg(Yeast) 860 EUR

Recombinant Octopus vulgaris Sperm protamine P5

MBS1170733-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 890 EUR

Recombinant Octopus vulgaris Sperm protamine P5

MBS1170733-005mgEColi MyBiosource 0.05mg(E-Coli) 510 EUR

Recombinant Octopus vulgaris Sperm protamine P5

MBS1170733-005mgYeast MyBiosource 0.05mg(Yeast) 710 EUR

Recombinant Octopus vulgaris Sperm protamine P5

MBS1170733-02mgEColi MyBiosource 0.2mg(E-Coli) 670 EUR

Recombinant Octopus vulgaris Sperm protamine P5

MBS1170733-05mgEColi MyBiosource 0.5mg(E-Coli) 715 EUR

Recombinant Octopus vulgaris Sperm protamine P4

MBS1063646-005mgBaculovirus MyBiosource 0.05mg(Baculovirus) 890 EUR

Recombinant Octopus vulgaris Sperm protamine P4

MBS1063646-005mgEColi MyBiosource 0.05mg(E-Coli) 510 EUR

Recombinant Octopus vulgaris Sperm protamine P4

MBS1063646-005mgYeast MyBiosource 0.05mg(Yeast) 705 EUR

Recombinant Octopus vulgaris Sperm protamine P4

MBS1063646-02mgEColi MyBiosource 0.2mg(E-Coli) 670 EUR

Recombinant Octopus vulgaris Sperm protamine P4

MBS1063646-05mgEColi MyBiosource 0.5mg(E-Coli) 710 EUR

Recombinant Octopus vulgaris Tubulin alpha chain

MBS1117659-002mgBaculovirus MyBiosource 0.02mg(Baculovirus) 1125 EUR

Recombinant Octopus vulgaris Tubulin alpha chain

MBS1117659-002mgEColi MyBiosource 0.02mg(E-Coli) 750 EUR

Recombinant Octopus vulgaris Tubulin alpha chain

MBS1117659-002mgYeast MyBiosource 0.02mg(Yeast) 910 EUR

Recombinant Octopus vulgaris Tubulin alpha chain

MBS1117659-01mgEColi MyBiosource 0.1mg(E-Coli) 900 EUR

Recombinant Octopus vulgaris Tubulin alpha chain

MBS1117659-01mgYeast MyBiosource 0.1mg(Yeast) 1065 EUR

Recombinant Octopus vulgaris S-crystallin 3 (OCTS3)

MBS1472408-002mgBaculovirus MyBiosource 0.02mg(Baculovirus) 1135 EUR

Recombinant Octopus vulgaris S-crystallin 3 (OCTS3)

MBS1472408-002mgEColi MyBiosource 0.02mg(E-Coli) 720 EUR

Recombinant Octopus vulgaris S-crystallin 3 (OCTS3)

MBS1472408-002mgYeast MyBiosource 0.02mg(Yeast) 885 EUR

Stereospecyficzność dehydrogenazy oktopinowej z guza żółciowego korony i dehydrogenazy oktopinowej z przegrzebków (maksimum Pectena) w odniesieniu do utleniania C-4 pierścienia dihydronikotynamidowego NADH badano poprzez określenie rozkładu radioaktywności po utlenieniu (4R)- i (4S)-[4-3H]NADH. Dehydrogenaza oktopinowa z guza koronowego i przegrzebków stereospecyficznie usuwa atom wodoru pro-S pierścienia dihydronikotynamidu z przeniesieniem znacznika do rozpuszczalnika i do produktu oktopiny (N-2-(1-karboksyetylo)-L-arginina). Chociaż w mniejszym stopniu, wymiana znacznika z (4S)-[4-3H]NADH z rozpuszczalnikiem wystąpiła, gdy dehydrogenaza oktopinowa z guza żółciowego korony lub z przegrzebków była inkubowana bez innych substratów. Omówiono możliwe mechanizmy wyjaśniające tę wymianę.

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